Fitness, risk taking, and spatial behavior covary with boldness in experimental vole populations

Abstract Individuals of a population may vary along a pace‐of‐life syndrome from highly fecund, short‐lived, bold, dispersive “fast” types at one end of the spectrum to less fecund, long‐lived, shy, plastic “slow” types at the other end. Risk‐taking behavior might mediate the underlying life history trade‐off, but empirical evidence supporting this hypothesis is still ambiguous. Using experimentally created populations of common voles (Microtus arvalis)—a species with distinct seasonal life history trajectories—we aimed to test whether individual differences in boldness behavior covary with risk taking, space use, and fitness. We quantified risk taking, space use (via automated tracking), survival, and reproductive success (via genetic parentage analysis) in 8 to 14 experimental, mixed‐sex populations of 113 common voles of known boldness type in large grassland enclosures over a significant part of their adult life span and two reproductive events. Populations were assorted to contain extreme boldness types (bold or shy) of both sexes. Bolder individuals took more risks than shyer ones, which did not affect survival. Bolder males but not females produced more offspring than shy conspecifics. Daily home range and core area sizes, based on 95% and 50% Kernel density estimates (20 ± 10 per individual, n = 54 individuals), were highly repeatable over time. Individual space use unfolded differently for sex‐boldness type combinations over the course of the experiment. While day ranges decreased for shy females, they increased for bold females and all males. Space use trajectories may, hence, indicate differences in coping styles when confronted with a novel social and physical environment. Thus, interindividual differences in boldness predict risk taking under near‐natural conditions and have consequences for fitness in males, which have a higher reproductive potential than females. Given extreme inter‐ and intra‐annual fluctuations in population density in the study species and its short life span, density‐dependent fluctuating selection operating differently on the sexes might maintain (co)variation in boldness, risk taking, and pace‐of‐life.


| INTRODUC TI ON
Risk-reward trade-offs may favor the coexistence of different behavioral types in populations (Sih & Del Giudice, 2012). Bolder animals may be rewarded for taking higher risks by producing more offspring, and shyer animals may have an increased life span but lower reproductive output per time unit. In species that are highly depredated, however, the fitness gains must clearly outweigh the survival costs of boldness to maintain bold behavior. Alternatively, different behavioral phenotypes can be favored under different environmental conditions, which may lead to similar fitness between behavioral phenotypes and fluctuations of phenotype frequencies in populations (Bergeron et al., 2013;Dingemanse et al., 2004;Nicolaus et al., 2016;Roth et al., 2021).
Similarly, selection related to fluctuations in population density can maintain variation in life history trajectories (Saether et al., 2016), linking ecological dynamics to evolutionary processes. Life history trajectories may be related to favorable physiological and behavioral phenotypes, forming an extended pace-of-life syndrome (POLS, e.g., Careau et al., 2008;Dammhahn et al., 2018;Réale et al., 2010) with fast POL individuals increasing their fitness by higher risk taking, and slow POL individuals by avoiding risks (Wolf et al., 2007a;Wolf et al., 2007b;Wright et al., 2019). Thus, both variation in life histories and the associated among-individual differences in behavior could potentially be explained through their eco-evolutionary dynamics with fluctuations in population density (Milles et al., 2022;Wright et al., 2019), group size, or composition of personality in groups (Roth et al., 2019).
Small rodents offer a suitable study system to assess whether and how among-individual differences translate into variation in risk taking and space use and have consequences for fitness components. Despite extreme predation pressure (Norrdahl & Korpimäki, 1998), consistent individual differences in risk taking have been observed in several small rodent species Lantová et al., 2011;Mazza et al., 2018). Small mammals in temperate environments follow very distinct life history trajectories within populations, with some individuals-born early or in the middle of the productive seasonreproducing immediately and repeatedly in the season of birth and other individuals-born late in the productive season-having to delay maturity, survive the unproductive season and, wait for the next productive season . These trajectories are flexible and triggered by density-dependent processes (Prévot-Julliard et al., 1999), allowing the parallel existence of very different life history trajectories, possibly connected to behavioral differentiation into pace-of-life syndromes and maintained by frequency-dependent selection during density fluctuations (Wright et al., 2019).
Consistent among-individual differences in behavior may contribute to variation in individual spatiotemporal distribution and might, thus, influence individuals' interactions with biotic and abiotic components of their environment (Bolnick et al., 2011;Wolf & Weissing, 2010). Whether feedback between space use and individual differences in behavior exists and how this potential feedback drives and/or maintains intraspecific (co)variation in these traits under heterogeneous environmental conditions is matter of current debate (Spiegel et al., 2017). In order to start illuminating these aspects, we need studies quantifying among-individual differences in behavior and space use independently from each other (e.g., birds; Arvidsson et al., 2017). More ideally, proxies of fitness components, such as reproductive success and survival, would allow assessing the consequences of interindividual differences in behavior. The main aim of this study was to investigate whether between-individual differences in risk taking and activity behavior that are measured in the laboratory are linked to space use in the field. The second aim was to investigate whether the laboratory measurements can be used to predict survival and reproductive success under field conditions.
To quantify among-individual variation in two behavioral traits, we conducted two repeated laboratory tests. We measured boldness and activity (Réale et al., 2007), which are highly positively correlated at the phenotypic level in common voles; that is, bolder individuals are more active Gracceva et al., 2014;Lantová et al., 2011). Subsequently, we ecologically validated these personality traits by quantifying space use in a grassland, the natural habitat of common voles, and tested the consequences of among-individual differences on survival and reproductive success in large outdoor enclosures in experimental populations, which also provided a social environment to the animals.
Space use of animals was monitored with automated radio telemetry (ART, e.g., Hoffmann et al., 2018;Kays et al., 2011;Schirmer et al., 2019), and risk taking via radio frequency identification (RFID) systems placed at risky locations. The combination of different methods

T A X O N O M Y C L A S S I F I C A T I O N
Behavioural ecology; Movement ecology should allow to complement their respective limitations in temporal or spatial accuracy and detection biases. We tested for relationships between among-individual differences in boldness on differences in survival probability, risk taking, space use, and reproductive success over 5 weeks under near-natural conditions. The study period of 5 weeks covers a substantial proportion of an average adult vole's life span of weeks to months (Halle & Stenseth, 2000).
We predicted that individual differences in boldness and activity, quantified in standardized tests in the laboratory, translate into behavioral differences in space use and risk taking under near-natural conditions. Specifically, we predicted that bold/active individuals occupy larger home ranges and core areas than shy/inactive individuals, as shown for other taxa including birds (Minderman et al., 2010) and small mammals (Boon et al., 2008;Schirmer et al., 2020).
Further, we predicted that bold/active individuals-in contrast to shy/inactive individuals-use unsafe open areas at the edge of the suitable habitat patches in large outdoor enclosures (i) with a higher propensity, (ii) a higher frequency, and (iii) longer duration because boldness predicts risk taking (Dammhahn & Almeling, 2012) and dispersal propensity (Cooper et al., 2017) in other small mammals.
We further expected lower survival of bold/active individuals compared to shy/inactive conspecifics because high levels of risk taking and activity may lead to increased predation (meta-analysis: Smith & Blumstein, 2008; but see Moiron et al., 2020). Elevated exploration and activity might pose a high predation risk, but may result in more encounters with the other sex, or increase attractiveness, and, thus, result in reproductive gains (Ophir et al., 2008;Smith & Blumstein, 2008;Sih et al., 2014; but see Araya-Ajoy et al., 2016).
Contrarily, high activity levels could also be advantageous if they are connected to the speed of exploration, like in eastern chipmunks (Tamias striatus) where fast explorers had lower mortality compared to slow explorers, probably because they had increased information about the environment (Bergeron et al., 2013). Our study period included two reproductive cycles of common voles, and we quantified the number of offspring produced during these cycles via genetic parentage analysis. We expected bold/active males to sire more offspring than shy/inactive males. Similarly, we expected bold/ active females to have higher reproductive success because they might occupy larger (or better quality) ranges (Schirmer et al., 2019).
Moreover, since boldness and exploration correlate in common voles , bold/active females might provision more food to their offspring than shy/inactive individuals as shown for more explorative blue tit (Mutzel et al., 2013).

| Overall study design
Our study had three steps (Table 1)  All captured individuals were subjected to a battery of repeated behavioral tests to assess the correlational structure of behavioral variables and temporal consistency of among-individual differences, that is, animal personality; these results are presented elsewhere  combined scores for activity and boldness were correlated at the phenotypic level, across all animals tested in the laboratory, male voles were more active than females but sexes did not differ in latencies. The individual time in captivity before the first behavioral test was performed did not explain variation in boldness nor activity (R 2 < .03, for all four variables).
Behavioral testing started 3-6 weeks after the animals were captured (to ensure that females were not pregnant, and that pregnant females were able to give birth, raise, and wean the litter), and as soon as a cohort of 24 animals had been collected (12 individuals per sex Across all test cohorts, absolute values of behavioral variables from individuals classified as bold (n = 56) differed from those of individuals classified as shy (n = 56 + 1 one additional shy animal released to the first enclosure by mistake, Student's t tests for all behavioral variables 2.4 < t < 7.8; all p < .02, Tables S1 and S2). Also body weights differed among types, with animals classified as shy being 10% heavier than animals classified as bold (t < 2.0, p < .049, Table S1). Vegetation along the inside of the enclosure walls was mowed in a strip of 1.5 m width to prevent animals from climbing ( Figure 1a).

| (b) Experimental populations in large nearnatural grassland enclosures
The strip was kept short by regular hand mowing, and intervals depended on local vegetation height and rain patterns. Mowing did not kill any of the collared voles. In the mowed area, the perceived avian predation risk is high for a ground dwelling mammal (Jacob & Brown, 2000).

| (c) Behavior in the field
Before the release into grassland enclosures, each animal was marked with a unique passive integrated transponder (PIT; 0.1g; Trovan ID-100, Euro ID, Germany), placed subcutaneously in the scapular region, for individual recognition at RFID readers. Animals were fitted with a radio telemetry collar (Biotrack, UK; 1.0 g including cable tie, <5% of mean body mass before release) with an individual radio frequency.
We placed barriers (Figure 1b Males were released two days prior to females to display potential differences in exploration behavior and avoid an immediate associate with locations of females (Ims, 1988). min-max range: 3-35 day ranges). We conducted calibration, precision, and maintenance checks on the telemetry grids before, in the middle, and after each replicate, using stationary experimental tags.
Calibrations lasted ca. 1 h leaving sufficient time to collect locations F I G U R E 1 Schematic display of one near-natural grassland enclosure. (a) Enclosures were equipped with Automated Radio Telemetry (ART) with (1) eight radio telemetry antennae (two in each corner) as part of an automated radio telemetry system (ART), (2) four guided passages with RFID readers (gray circles) in the vegetation free area strip (white area) along the enclosure wall, and (3)  We calculated day ranges (i.e., 95% density Kernels) and core area of day ranges (i.e., 50% density Kernels) as estimates of individual vole movement and mobility (Worton, 1987(Worton, , 1995. Since all analyses conducted with both kernel sizes yielded very similar statistics (both were based on the same location data set), we present only the statistical results on day ranges here.
We hypothesized that behavior in the field also depends on the social interactions in the experimental population. Particularly, mobility of males may vary with the availability of mating partners, which would be low during synchronized pregnancy phases of females. Further, we hypothesized that boldness types may cope differently with being released to the unknown habitat (Veerbek et al., 1994). Therefore, we included the following experimental phases based on the species' life history into the analysis of location data (Table 1)

| (d) Fitness
To remove adults and their offspring from the enclosures, we set live traps after 35 days (Table 1). It took up to 5 days until animals were removed. Individuals without a mobile radio tracking signal that were not recaptured during removal trapping or did not reappear in a later replicate (6 cases) were considered to be dead. To estimate reproductive success, we collected small tissue samples from the ears of adult voles before release to the enclosures, from offspring born in and captured from the enclosures (N = 335 juveniles), and from offspring born to females kept in singe cages after the experiment (N = 85 juveniles). Laboratory procedures for genotyping followed Braaker and Heckel (2009 ; Table S3). Microsatellite alleles were determined using GeneMapper ® Software, version 3.7 (Applied Biosystems). The number of alleles ranged between two and 32 (mean = 13.9) per microsatellite locus. We used the software CERVUS 3.0.3 (Kalinowski et al., 2007;Marshall et al., 1998)  There were six animals among those candidates where no offspring was assigned, but which had been able to potentially sire offspring, as indicated either by their recapture after the experiment (n = 3) or the polyphasic activity signature of their radio signals (n = 3, see supplemental material for exemplary diagnostic plots).

| (e) Statistical analyses
To test whether boldness type explained variation in risk taking, space use, survival, and reproductive success, we used linear or generalized linear mixed effects models (LMM or GLMM) run with the R package "lme4" (Bates et al., 2015). The underlying error distributions were specified as binomial for probabilities (survival, reproduction, visits of risky areas), as Poisson for count variables (number of visits of risky areas, number of offspring) and as Gaussian for continuous variables (home range size, core area size, duration of visits of risky areas). Given a biased distribution as based on visual inspection, we log-transformed the duration of visits. Models included our predictor boldness type (shy or bold) and sex (male or female) as fixed effects, and their interaction. Further, we included control variables as fixed effects into initial models: the starting month of the replicate (to control for seasonal variation as covariate) as a continuous covariate, and the experimental phase (with four levels, see Table 1 for details) for models on space use only. Experimental phase was specified in interaction with boldness type and sex because we expected space use to vary with the phases of the experiment and in particular with female reproductive activity in our artificially reproductively synchronized populations. As random effect, we included population replicate ID (specified as random intercept) to control for potential variation among replicates such as vegetation height, rain events, or predation pressure. Such external properties could potentially affect the behavior of the entire population. Furthermore, vegetation and weather may affect the quality of our tracking calibration, the recapture success. For exploration of the data, we experimented with different random structures (e.g., including the identity of the enclosure or adding the year as a fixed factor, but population ID as random factor captured this variation). In models of space use and risk taking, we had repeated measurements of individuals and therefore added individual ID as a second random effect to the mixed models.
We assessed model fit visually based on inspections of residual   Table 2) and visitation probability of an animal was independent of its boldness (χ 2 = 0.4, p = .524). Among visiting animals males visited more often (7.4 ± 10 visits) than females (3.5 ± 6.2 visits, χ 2 = 14.1, p < .001, Figure 2b), and bold individuals more often (8.3 ± 11.9) than shy individuals (4.7 ± 6.1 visits, χ 2 = 22.3, p < .001), and the number of visits was higher in replicates later in the season (month: χ 2 = 4.8, p = .029, Table 2). The duration of a visit was highly repeatable within individuals (R = .632, CI 0.50-0.73) and was depending on an interaction of experimental phase and sex (χ 2 = 17.53, p < .001, Table 3, Figure 3a). Visited risky areas for shorter periods during the first pregnancy phase (Grav 1) compared with the exploration phase (Expl) and the second pregnancy (Grav 2). In males, we detected no effects of experimental phase or boldness type.

| Fitness of animals
In total, 73 of 113 (65%) released common voles were recaptured from the enclosures. Females tended to survive better (73%, Note: Shown are model estimates of GLMMs based on different sample sizes of animals (n, given for each model) and different numbers of populations, which were included as a random factor. Covariate and interaction were removed if p > .1. The reference levels for categorical predictors are bold (for boldness type) and female (for sex). Shown are estimates (β) and their standard errors (SE), z-values and p-values as well as R 2 marginal as the variance explained by fixed factors, and R 2 conditonal as the variance explained by fixed and random factors. Significant effects are marked with bold font.

| DISCUSS ION
In experimentally created populations of known behavioral-type composition, we were able to show that individual differences in boldness behavior covaried with risk taking, space use, and fitness under near natural conditions. We created experimental populations combining the opposite ends of the distribution of behavioral phenotypes (bold and shy animals) and studied behavior in the field with automated tracking methods. We found that behaviors measured in the field were consistent within individuals over time, quantifying themselves for animal personality traits. We further found that bold animals of both sexes visited the risky edges of the enclosures more frequently than shy animals of the same sex. In females, effects of boldness type were detected during limited times only (Figure 4).
While males ranged over larger areas than females (as shown earlier for this species, e.g., Briner et al., 2005), range sizes differed between shy and bold females immediately upon release. Shy females apparently explored larger areas initially and then settled in smaller ranges, while the opposite pattern was observed in bold females and males of both behavioral phenotypes. Bolder males took higher risks and fathered more offspring than shy males. Boldness did not explain survival probability in both sexes, however. Mortality of voles tended to increase in autumn (Figure 2a), probably due to colder weather and decreasing quality of forage, mirroring annual population dynamics of common voles .

| Behavior and boldness types
Boldness as measured in many small mammals in laboratory settings using open field and exploration tasks may be a direct predictor of Note: Risk taking was recorded with RFID readers in risky, short grass areas of enclosures. Range sizes were recorded with automated radio tracking. Random structure corrects for repeats within populations of up to 8 animals, and repeated measures within individuals. Both marginal and conditional R 2 for risk taking R 2 = .07, for day ranges marginal R 2 = .08, conditional R 2 = .49. Significant effects are marked with bold font.

TA B L E 3
Estimates (and their standard error, SE) of effect sizes of sex, boldness type, and experimental phase on behavior of common voles recorded in large grassland enclosures (2500 m 2 ) analyzed with linear mixed models. BNT: Boldness type risk taking (Dammhahn & Almeling, 2012). The main source of mortality for voles is predation, rendering them a key species in natural food chains with many ground and avian predators preying on them (Halle, 1988;Jędrzejewski & Jędrzejewska, 1993;Norrdahl & Korpimӓki, 1995). Since boldness may be directly linked to mortality risk (Smith & Blumstein, 2008, but see Moiron et al., 2020), it should have a strong impact on spatial behavior, and the exposure to predators.
In the experimental populations in our study, bold males were taking higher risks by visiting the short vegetation edges of the enclosure more frequently than shy males. Boldness is often correlated with exploration at the phenotypic level, so bolder individuals were often reported to explore an area faster than shy individuals, which could result in a more superficial exploration and exploitation of resources (Mazza et al., 2018;Sih et al., 2004;Wolf et al., 2007a;Wolf et al., 2007b). In our study, we found some support for this pattern with bold males being registered in the risky area of the enclosure more often and for shorter periods than shy males, probably indicating a quicker and superficial exploration of these areas.
The duration of single visits did not differ among males, but among females. Females visited the risky edge less frequently than males, and stayed very shortly at the passage counters during their first pregnancy, compared to the exploration phase and the second pregnancy ( Figure 3). Since long stays indicated a slower and more careful passage, as indicated by our pilot experiment, we assume these phases are used for exploration (of the novel area, or to find a new nest for giving birth to the second litter), while during the first pregnancy females passed the counters quickly and on paths known to them. Females never appeared at the passage counters during the second mating phase, probably because voles mate briefly during with individual behavioral differences (Schirmer et al., 2020). Thus, overall among-individual differences in space use may contribute to individual niche specialization (Pearish et al., 2013;Spiegel et al., 2017), facilitating the coexistence of similar species (Schirmer et al., 2020). The distribution of individuals in space and time is an important determinant for key aspects of the social system, for example, the mating system (Heckel & von Helversen, 2002, 2003Lukas & Clutton-Brock, 2013), and of foraging under risk. Hence, behavioral type-specific space use should have consequences for survival and reproductive success.
Directly after transfer to the novel environment (exploration phase), bold females and both types of males in our experimental populations used smaller day ranges than at later stages of the experiment, indicating an initial reduction in mobility. Shy females used larger areas during the first three days, and settled in areas that later allowed them to maintain small home ranges ( Figure S1).
At first glance, this differs from established populations in different species where bold animals (of both sexes) had larger ranges than shy ones (rodents: Boon et al., 2008;Schirmer et al., 2020, birds: Minderman et al., 2010. Our finding is more in line with observations of shy animals being more thorough explorers in novel environments (Marchetti & Drent, 2000;Mazza et al., 2018;Mutzel et al., 2013;Veerbek et al., 1994), which might give them an advantage under changing and harsh environmental conditions.
(1) Bolder males might take higher risks in roaming in space to find receptive females and/or defend receptive females more successfully to monopolize paternity (Ophir et al., 2008;Smith & Blumstein, 2008;Wolf et al., 2007a;Wolf et al., 2007b); indeed, in our study bolder males were detected more often in risky areas of the enclosures. Appearance in such areas is sometimes used to infer dispersal tendencies (Hahne et al., 2011) and may also indicate wider roaming areas (Schirmer et al., 2020;Ward-Fear et al., 2018).
(2) Females could have a preference for bolder males (Godin & Dugatkin, 1996). If boldness was selected for in males, we would expect males to generally be bolder than females (Schuett et al., 2010), which is not supported by our other studies on voles success of males may be primarily determined by dominance rank (Dewsbury, 1982;Ellis, 1995) rather than personality per se, but both traits can be highly entangled so that boldness may predict dominance. (4) Among-individual variation in behavior could be part of a larger pace-of-life syndrome Réale et al., 2010) and covariation between these traits might be maintained by density-dependent selection (Milles et al., 2022;Wright et al., 2019).
Microtine voles, in most places, frequently and predictably undergo massive fluctuations in population density, which are accompanied by population-level differences in behavioral type ) and social environmental conditions. Further, for short-lived iteroparous animals in seasonally fluctuating environments, life history trajectories and social environmental conditions (e.g., density) are predictable (Eccard et al., 2017;. Selection may favor bolder behavioral types in high density and high We did not detect an effect of boldness type on reproductive success in females. Thus, the fitness consequences of boldness might be sex-specific, similar to the findings in black browed albatrosses (Patrick & Weimerskirch, 2014). Access to food and safety are major determinants of reproductive success in female mammals (Crook & Gartlan, 1966;Emlen & Oring, 1977;Lukas & Clutton-Brock, 2013;Ophir et al., 2008;Terborgh & Janson, 1986); since common voles mainly eat grass and find shelter in underground burrows, our large grassland enclosures should not have provided a resource limited environment. Further, reproductive skew is generally lower in females than in males (Bateman, 1948) and once female voles reproduce, they usually produce entire litters. We expected bold females to occupy larger home ranges (as bank voles under natural conditions: Schirmer et al., 2019) and thus be able to provision their offspring better (e.g., as in blue tits: Mutzel et al., 2013) compared to shy females. However, differences in provisioning (lactation) would be difficult to detect among different types of female mammals in an outdoor study.
In contrast to our prediction but in line with results of a recent meta-analysis (Moiron et al., 2020), survival did not differ between boldness types. The finding may be caused by the rather benign setting of our experiment in a favorable season, a low population density, and reduced predation pressure since ground predators were excluded. Alternatively, limited space might be another explanation, since male voles might roam larger areas under natural conditions than offered in our enclosures. Overall, the survival rate of common voles in our study (35% over 7 weeks) seemed high compared to those reported elsewhere: 2 to 9% daily mortality of voles with radio transmitters (field voles, East European voles and bank voles; Norrdahl & Korpimӓki, 1995), or 50% mortality over four weeks in agricultural fields (common voles;Jacob, 2003). In our experiment, survival dropped toward the end of the season (Figure 2a) for animals of any boldness type, when in wild populations peak densities would crash

| CON CLUS IONS
Overall, our results highlight that among-individual differences in behavior translate into variation in space use, risk taking, and reproductive success in near-natural populations. Reproduction was biased toward single bold males in late summer replicates. Since variation in boldness is maintained in natural populations, we assume that shy types may have fitness advantages in other seasons (Lonn et al., 2017) or at different population densities (Wright et al., 2019), which remains to be tested. With daily range sizes being highly repeatable within individuals, consistent individual space use patterns may facilitate individual niche specialization and thus affect within-and between-species ecological interactions. We show with this experiment, that behavioral phenotypes covary with risk-taking behavior in the field, and that behavioral differences are thus expressed in natural settings. We can further show that behavioral phenotypes are fitness relevant.

ACK N OWLED G M ENT
We thank all students and student helpers who trapped and ob-